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Co-culture of primary CLL cells with bone marrow mesenchymal cells, CD40 ligand and CpG ODN promotes proliferation of chemoresistant CLL cells phenotypically comparable to those proliferating in vivo.

Purroy N, Abrisqueta P, Carabia J, Carpio C, Palacio C, Bosch F, Crespo M - Oncotarget (2015)

Bottom Line: Importantly, co-cultured primary CLL cells shared many phenotypical features with circulating proliferative CLL cells, such as upregulation of ZAP-70 and CD38 and higher CD49d and CD62L expression.This indicates aggressiveness and capability to interact with surrounding cells, respectively.In addition, levels of CXCR4 were decreased due to CXCR4 internalization after CXCL12 stimulation by BM stromal cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Experimental Hematology, Department of Hematology, Vall d'Hebron University Hospital, Universitat Autònoma de Barcelona, Barcelona, Spain.

ABSTRACT
Chronic lymphocytic leukemia (CLL) cells residing in the bone marrow (BM) and in secondary lymphoid tissues receive survival and proliferative signals from the microenvironment, resulting in persistence of residual disease after treatment. In this study, we characterized primary CLL cells cultured with BM stromal cells, CD40 ligand and CpG ODN to partially mimic the microenvironment in the proliferative centers. This co-culture system induced proliferation and chemoresistance in primary CLL cells. Importantly, co-cultured primary CLL cells shared many phenotypical features with circulating proliferative CLL cells, such as upregulation of ZAP-70 and CD38 and higher CD49d and CD62L expression. This indicates aggressiveness and capability to interact with surrounding cells, respectively. In addition, levels of CXCR4 were decreased due to CXCR4 internalization after CXCL12 stimulation by BM stromal cells. We suggest that this co-culture system can be used to test drugs and their combinations that target the proliferative and drug resistant CLL cells.

No MeSH data available.


Related in: MedlinePlus

The co-culture of primary CLL cells with BMSC, CD40L and CpG ODN promotes an immunophenotype comparable to that from proliferating CLL cells found in PB(A) PBMC from 40 patients diagnosed with CLL were used to analyze by FC the differential expression of CD38, CD49d, CD62L, CXCR4, CXCR5 and CCR7 in Ki-67-negative vs. positive CLL cells. (B) Primary CLL cells from 12 patients were cultured in suspension or in co-culture with BMSC, CD40L and CpG ODN for 48 hours and the expression ofCD38, CD49d, CD62L, CXCR4, CXCR5 and CCR7 were analyzed. (C) PBMC from 10 patients diagnosed with CLL were cultured in suspension or in co-culture with BMSC, CD40L and CpG ODN for 48 hours and the percentage of T cells and their expression levels of Ki-67, CD38 and CD69 were analyzed. (*P<0.05, **P<0.01, ***P<0.001, ns: non significant, paired T-test).
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Figure 2: The co-culture of primary CLL cells with BMSC, CD40L and CpG ODN promotes an immunophenotype comparable to that from proliferating CLL cells found in PB(A) PBMC from 40 patients diagnosed with CLL were used to analyze by FC the differential expression of CD38, CD49d, CD62L, CXCR4, CXCR5 and CCR7 in Ki-67-negative vs. positive CLL cells. (B) Primary CLL cells from 12 patients were cultured in suspension or in co-culture with BMSC, CD40L and CpG ODN for 48 hours and the expression ofCD38, CD49d, CD62L, CXCR4, CXCR5 and CCR7 were analyzed. (C) PBMC from 10 patients diagnosed with CLL were cultured in suspension or in co-culture with BMSC, CD40L and CpG ODN for 48 hours and the percentage of T cells and their expression levels of Ki-67, CD38 and CD69 were analyzed. (*P<0.05, **P<0.01, ***P<0.001, ns: non significant, paired T-test).

Mentions: With the aim of comparing primary CLL cells cultured ex vivo in conditions mimicking the microenvironment found in the proliferative centers with proliferating subclones of CLL cells found in PB from patients with active disease, we initially analyzed proliferating CLL cells from 40 patients diagnosed with CLL. For this, we analyzed by FC the differential expression of CD38, CD49d, CD62L and the chemokine receptors CXCR4, CXCR5 and CCR7 in Ki-67 positive vs. Ki-67 negative CLL cells (Figure 2A). Mean percentage of Ki-67 expression in CLL samples was 1.40±0.26 (range, 0.05-7.41). Ki-67-positive CLL cells showed higher expression levels of CD38 (mean MFI of CD38 expression in Ki-67 positive cells vs. Ki-67 negative cells: 57.46±8.43 vs. 25.41±3.83, P<0.001). The expression levels of integrin CD49d and selectin CD62L were also higher in Ki-67 positive CLL cells than in Ki-67 negative cells (mean MFI of CD49d expression in Ki-67 positive cells vs. Ki-67 negative cells: 44.92±10.15 vs. 39.53±10.43, P<0.01; mean MFI of CD62L expression in Ki-67 positive cells vs. Ki-67 negative cells: 37.87±10.03 vs. 31.48±9.41, P<0.05) while the expression levels of all the chemokine receptors analyzed were significantly lower (mean MFI of CXCR4 expression in Ki-67 positive cells vs. Ki-67 negative cells: 172.3±20.03 vs. 223.2±22.35, P<0.001; mean MFI of CXCR5 expression in Ki-67 positive cells vs. Ki-67 negative cells: 343.4±31.37 vs. 428.7±38.18, P<0.001; mean MFI of CCR7 expression in Ki-67 positive cells vs. Ki-67 negative cells: 110.1±8.07 vs. 149.2±10.65, P<0.001)(Figure 2A).


Co-culture of primary CLL cells with bone marrow mesenchymal cells, CD40 ligand and CpG ODN promotes proliferation of chemoresistant CLL cells phenotypically comparable to those proliferating in vivo.

Purroy N, Abrisqueta P, Carabia J, Carpio C, Palacio C, Bosch F, Crespo M - Oncotarget (2015)

The co-culture of primary CLL cells with BMSC, CD40L and CpG ODN promotes an immunophenotype comparable to that from proliferating CLL cells found in PB(A) PBMC from 40 patients diagnosed with CLL were used to analyze by FC the differential expression of CD38, CD49d, CD62L, CXCR4, CXCR5 and CCR7 in Ki-67-negative vs. positive CLL cells. (B) Primary CLL cells from 12 patients were cultured in suspension or in co-culture with BMSC, CD40L and CpG ODN for 48 hours and the expression ofCD38, CD49d, CD62L, CXCR4, CXCR5 and CCR7 were analyzed. (C) PBMC from 10 patients diagnosed with CLL were cultured in suspension or in co-culture with BMSC, CD40L and CpG ODN for 48 hours and the percentage of T cells and their expression levels of Ki-67, CD38 and CD69 were analyzed. (*P<0.05, **P<0.01, ***P<0.001, ns: non significant, paired T-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4480705&req=5

Figure 2: The co-culture of primary CLL cells with BMSC, CD40L and CpG ODN promotes an immunophenotype comparable to that from proliferating CLL cells found in PB(A) PBMC from 40 patients diagnosed with CLL were used to analyze by FC the differential expression of CD38, CD49d, CD62L, CXCR4, CXCR5 and CCR7 in Ki-67-negative vs. positive CLL cells. (B) Primary CLL cells from 12 patients were cultured in suspension or in co-culture with BMSC, CD40L and CpG ODN for 48 hours and the expression ofCD38, CD49d, CD62L, CXCR4, CXCR5 and CCR7 were analyzed. (C) PBMC from 10 patients diagnosed with CLL were cultured in suspension or in co-culture with BMSC, CD40L and CpG ODN for 48 hours and the percentage of T cells and their expression levels of Ki-67, CD38 and CD69 were analyzed. (*P<0.05, **P<0.01, ***P<0.001, ns: non significant, paired T-test).
Mentions: With the aim of comparing primary CLL cells cultured ex vivo in conditions mimicking the microenvironment found in the proliferative centers with proliferating subclones of CLL cells found in PB from patients with active disease, we initially analyzed proliferating CLL cells from 40 patients diagnosed with CLL. For this, we analyzed by FC the differential expression of CD38, CD49d, CD62L and the chemokine receptors CXCR4, CXCR5 and CCR7 in Ki-67 positive vs. Ki-67 negative CLL cells (Figure 2A). Mean percentage of Ki-67 expression in CLL samples was 1.40±0.26 (range, 0.05-7.41). Ki-67-positive CLL cells showed higher expression levels of CD38 (mean MFI of CD38 expression in Ki-67 positive cells vs. Ki-67 negative cells: 57.46±8.43 vs. 25.41±3.83, P<0.001). The expression levels of integrin CD49d and selectin CD62L were also higher in Ki-67 positive CLL cells than in Ki-67 negative cells (mean MFI of CD49d expression in Ki-67 positive cells vs. Ki-67 negative cells: 44.92±10.15 vs. 39.53±10.43, P<0.01; mean MFI of CD62L expression in Ki-67 positive cells vs. Ki-67 negative cells: 37.87±10.03 vs. 31.48±9.41, P<0.05) while the expression levels of all the chemokine receptors analyzed were significantly lower (mean MFI of CXCR4 expression in Ki-67 positive cells vs. Ki-67 negative cells: 172.3±20.03 vs. 223.2±22.35, P<0.001; mean MFI of CXCR5 expression in Ki-67 positive cells vs. Ki-67 negative cells: 343.4±31.37 vs. 428.7±38.18, P<0.001; mean MFI of CCR7 expression in Ki-67 positive cells vs. Ki-67 negative cells: 110.1±8.07 vs. 149.2±10.65, P<0.001)(Figure 2A).

Bottom Line: Importantly, co-cultured primary CLL cells shared many phenotypical features with circulating proliferative CLL cells, such as upregulation of ZAP-70 and CD38 and higher CD49d and CD62L expression.This indicates aggressiveness and capability to interact with surrounding cells, respectively.In addition, levels of CXCR4 were decreased due to CXCR4 internalization after CXCL12 stimulation by BM stromal cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Experimental Hematology, Department of Hematology, Vall d'Hebron University Hospital, Universitat Autònoma de Barcelona, Barcelona, Spain.

ABSTRACT
Chronic lymphocytic leukemia (CLL) cells residing in the bone marrow (BM) and in secondary lymphoid tissues receive survival and proliferative signals from the microenvironment, resulting in persistence of residual disease after treatment. In this study, we characterized primary CLL cells cultured with BM stromal cells, CD40 ligand and CpG ODN to partially mimic the microenvironment in the proliferative centers. This co-culture system induced proliferation and chemoresistance in primary CLL cells. Importantly, co-cultured primary CLL cells shared many phenotypical features with circulating proliferative CLL cells, such as upregulation of ZAP-70 and CD38 and higher CD49d and CD62L expression. This indicates aggressiveness and capability to interact with surrounding cells, respectively. In addition, levels of CXCR4 were decreased due to CXCR4 internalization after CXCL12 stimulation by BM stromal cells. We suggest that this co-culture system can be used to test drugs and their combinations that target the proliferative and drug resistant CLL cells.

No MeSH data available.


Related in: MedlinePlus